Apoptosis plays an essential role in the development and maintenance of homeostasis within multicellular organisms. Impairment of apoptosis is not only central to cancer development but also renders tumors refractory to cytotoxic therapy. The BCL-2 family proteins, consisting of both anti-apoptotic and proapoptotic members, constitute a crucial checkpoint in the cell death pathway. Our prior studies have helped delineate the core apoptotic pathway in mammals. The BH3-only molecules activate multidomain proapoptotic BAX and BAK to releases cytochrome c for caspase activation and initiate caspase-independent mitochondrial dysfunction. Conversely, anti-apoptotic BCL-2/BCL-XL sequesters BH3-only molecules into inert complexes, thus preventing the activation of BAX and BAK. Genetic studies revealed that absence of proapoptotic BAX and BAK creates a profound block in apoptosis triggered by diverse death signals including cancer therapeutics. Thus, activation of a multidomain member, BAX or BAK, appears to be an obligate gateway to the mitochondrion-mediated cell death program. However, it remains unclear whether all the BH3-only molecules are capable of activating BAX and BAK, how the BH3-only molecules activate BAX and BAK, and which BH3-only molecule and how it is activated by each specific death signal to initiate the apoptotic signaling cascade. Emerging evidence suggests that anti-apoptotic BCL-2 members are selectively inactivated by different BH3-only molecules, but detailed functional map of individual BH3-only molecules in antagonizing anti-apoptotic BCL-2 members is still missing. Moreover, it remains debated whether anti- apoptotic BCL-2 members inhibit apoptosis through direct inhibition of BAX/BAK and/or sequestration of BH3-only molecules. Finally, it is critical to determine how BAX and BAK are kept in check in viable cells. Are they kept in check by anti-apoptotic BCL-2 members or by other associated proteins such as VDAC2? To address these questions, we propose to characterize the BH3-only molecules, assign and identify individual BH3-only molecules to integrate apoptotic signaling cascades and mitochondrion-dependent death program, dissect the mechanisms by which BH3-only molecules activate BAX and BAK, and establish a functional network of differential regulation among various BCL-2 subfamilies. Our ultimate goal is to translate the mechanisms governing death/survival decision by BCL-2 family into anti-cancer strategies.